PROMIS: Global Analysis of PROtein‐Metabolite Interactions

Abstract

Small molecules are not only intermediates of metabolism, but also play important roles in signaling and in controlling cellular metabolism, growth, and development. Although a few systematic studies have been conducted, the true extent of protein–small molecule interactions in biological systems remains unknown. PROtein–metabolite interactions using size separation (PROMIS) is a method for studying protein–small molecule interactions in a non‐targeted, proteome‐ and metabolome‐wide manner. This approach uses size‐exclusion chromatography followed by proteomics and metabolomics liquid chromatography–mass spectrometry analysis of the collected fractions. Assuming that small molecules bound to proteins would co‐fractionate together, we found numerous small molecules co‐eluting with proteins, strongly suggesting the formation of stable complexes. Using PROMIS, we identified known small molecule–protein complexes, such as between enzymes and cofactors, and also found novel interactions. © 2019 The Authors. Basic Protocol 1: Preparation of native cell lysate from plant material Support Protocol: Bradford assay to determine protein concentration Basic Protocol 2: Separation of molecular complexes using size‐exclusion chromatography Basic Protocol 3: Simultaneous extraction of proteins and metabolites using single‐step extraction protocol Basic Protocol 4: Metabolomics analysis Basic Protocol 5: Proteomics analysis

Global mapping of protein–metabolite interactions in Saccharomyces cerevisiae reveals that Ser-Leu dipeptide regulates phosphoglycerate kinase activity

“…The diploid, prototrophic YSBN2 strain in the logarithmic phase of growth was used as starting material. The overall experimental strategy included: (i) preparation of the native, soluble lysate, (ii) size fractionation of complexes using a size exclusion chromatography (SEC) and (iii) untargeted analysis of the complex components using mass spectrometry-based metabolomics and proteomics 22,23 (Fig. 1a).…”

“…To address this limitation, we have developed an approach, termed PROMIS, which enables a cell-wide analysis of the protein-metabolite and protein-protein interactomes 22,23 . Similar to the previously mentioned approaches, PROMIS starts with a native cellular lysate and thus operates in close to in vivo conditions.…”

Combination of network and molecule structure accurately predicts competitive inhibitory interactions

“…E. coli cells were harvested by centrifugation (RT, 4000g) and snap frozen in liquid nitrogen. PROMIS experiment was performed as described previously in [25] . Briefly, E. coli native lysate containing endogenous protein- protein and protein-metabolites complexes corresponding to 40 mg of protein, was separated using a Sepax SRT SEC-300 21.2 × 300 mm column (Sepax Technologies, Inc., Delaware Technology Park, separation range 1.2 mDa to 10 kDa) connected to an ÄKTA explorer 10 (GE Healthcare Life Science, Little Chalfont, UK).…”

“…Further proteins and metabolites from the lyophilized fractions were extracted using a methyl- tert -butyl ether (MTBE)/methanol/water method [26] , where, molecules are simultaneously separated into organic phase (lipids), aqueous phase (polar and semi-polar metabolites) and protein pellets. Aqueous phase and protein pellets were dried in a SpeedVac and subjected metabolomic and proteomic analysis as described by Sokolowska et al [25] .…”

“…Analysis of the metabolite and protein elution profiles was performed as describe before Sokolowska et al [25] , Gorka M [27] , and included data filtering, normalization and deconvolution. Obtained data were integrated together using Pearson correlation assuming that what correlates, and hence co-eluates, together is potentially in the complex.…”